The Function Of Differential Expression Of CircRNA In COPD Patients Induced By PM_2.5 Exposure

A large numbers of epidemiological studies have indicated the strong correlation between fine particulate matter(PM_2.5)exposure and respiratory diseases.circRNA is characterized by its high stability and tissue specificity.circRNA has also been proven to be involved in the occurrence and development of a variety of diseases.In this study,the circRNA Microarray technique was used to detect the expression of circRNA profile in plasma of COPD patients exposed to PM_2.5.qRT-PCR was used to verified the level of significally altered circRNA in COPD patients'plasma.Meanwhile,the models of chronic PM_2.5-exposed HBE cells in vitro and the model of mice exposed to PM_2.5 in vivo were established,which were used for further studies on circRNA function.In addition,different expression proteins in plasma exosomes of COPD patients exposed to PM_2.5 were detected by protein spectroscopy?iTRAQ?,aiming to explore the relationship between circRNA and COPD in the protein levels following by PM_2.5exposure.1.CircRNA expression profile changed in COPD patient plasma caused by PM2.51.CircRNA Microarray analysis was used to investigate the changes of circRNA expression in plasma of COPD patients exposed to PM_2.5.Microarray data showed that 21differentially expressed circRNA were found in COPD plasma,among which 15 circRNA were significantly up-regulated and 6 circRNA were significantly down-regulated?P<0.05?.2.The plasma of COPD patients exposed to PM_2.5 was collected from Nanjing Chest Hospital.The circRNA with significantly fold change were selected,including 4 up-regulated circRNA?hsa_circ₀061265,hsa_circ₀037516,hsa_circ₀001503 and hsa_circ₀005045?and 1 down-regulated circRNA?hsa_circ₀028173?.According to the result of qRT-PCR in 34COPD patients,the result showed that the expression of hsa_circ₀061265,hsa_circ₀037516,hsa_circ₀001503 and hsa_circ₀005045 were significantly up-regulated,and hsa_circ₀028173 were significantly down-regulated.These results were consistent with the circRNA microarray?P<0.05?.3.hsa_circ₀005045 was selected from the above verified circRNA with significantly fold change for further verification.qRT-PCR results showed that hsa_circ₀005045 expression was significantly up-regulated in plasma of 98 COPD patients exposed to PM_2.5?P<0.001?.2.Expression of hsa_circ₀005045 in vivo and in vitro1.The Circbase and UCSC databases showed that hsa_circ₀005045 is locates on the short arm of human chromosome 2,which is derived from RTN4 gene.The experiment of cytoplasmic separation showed that hsa_circ₀005045 was mainly concentrated in the cytoplasm.According to agarose gel electrophoresis and sanger sequencing results,hsa_circ₀005045 was formed in cytoplasm by reverse splicing of the second and third exon of RTN4 gene,and existed in plasma in the form of a ring,the splicing sites were G and T.2.A long-term exposure model of PM_2.5 to human bronchial epithelial cells?HBE?was established.The final concentrations were set to 0,100,and 500?g/ml.Cell morphology and proliferation of different algebraic cells were observed.3.CCK8 method was used to detect the proliferation of PM_2.5-exposed HBE cells?P9,P12,P15?.With the increase of passages,the cell proliferation ability of PM_2.5-exposed group were increased accordingly.Compared to the control group,the proliferation of HBE cells was significantly increased after 12 passages.Therefore,cells with long-term exposure of P15 were selected for subsequent cell and molecular tests?P<0.05?.4.HBE cells exposed to PM_2.5 for a long time?P15?were selected for qRT-PCR experiment,and the results showed that the level of hsa_circ₀005045 was significantly up-regulated after PM_2.5 exposure,and its maternal gene RTN4 expression was significantly down-regulated,compared to the control group?P<0.05?.5.The model of COPD-like changes in mice was established after PM_2.5 was intratracheal instilled to mice.Pathological changes of murine mice were observed after 7,14 and 28 days of PM_2.5-exposure.Compared to the control group?tracheal aspiration of PBS?,the result showed that different degrees of emphysema and alveolitis were observed in lung tissues of the mice after 7,14 and 28 days exposed?P<0.05?.6.Retrieved by the NCBI-blast database,it was found that 91%of the base sequences between human hsa_circ₀005045 and mouse mmu_circ₀002590 were matched.It was suggested that human and mouse circRNA were homologous.The results of agarose gel electrophoresis and sanger sequencing showed that mmu_circ₀002590 was also present in the plasma in the form of a ring,and the bases of its splicing sites were A and C.7.The expression level of mmu_circ₀002590 was further verified in the plasma of PM_2.5-exposed mice by qRT-PCR.The results showed that the expression of mmu_circ₀002590 was significantly up-regulated in 7 days,14 days and 28 days after PM_2.5 exposure?P<0.05?.3.Exploration of biological function of hsa_circ₀0050451.The protein spectrum?iTRAQ?,was used to determine the changes of protein profile in plasma exosomes of COPD patients exposed to PM_2.5.The results showed that there were 66differentially expressed proteins caused by PM_2.5 exposure,among which 30 were up-regulated and 36 down-regulated?FC=1.2,P<0.05?.The number of polypeptide fragments was concentrated in 7-13,and the protein mass was between 0-200 KDa.According to Protein-protein interaction?PPI?analysis,ELANE,PRDX2,and CA2 were up-regulated,while TTR,KNG1,and APOC3 were down-regulated proteins.These proteins played central roles in PPI network and provided clues for further research.2.ELANE and PRDX2 were selected to study the relationship between hsa_circ₀005045and COPD.ELISA results showed that the expression of ELANE and PRDX2 were significantly up-regulated in plasma of 16 COPD patients,which was consistent with the results of protein spectrum?P<0.05?.3.Western Blot experiments were conducted on HBE cells exposed to PM_2.5?P15?.The results showed that PRDX2 level was singnificantly increased in PM_2.5-exposed HBE cells?P<0.05?,while the ELANE expression was not observed in HBE cells.4.Immunohistochemical experiments were conducted on mice with COPD-like changes exposed to PM_2.5.Compared to the control group,the expression of PRDX2 and ELANE in lung tissues of mice exposed to PM_2.5 in 7 days,14 days and 28 days were significantly up-regulated?P<0.05?.5.The interaction between hsa_circ₀005045 and PRDX2 was predicted by the online software RPISeq.The scores for the potential binding between hsa_circ₀005045 and PRDX2were 0.55?RF?and 0.80?SVM?.The scores for the potential binding between hsa_circ₀037516and PRDX2 were 0.60?RF?and 0.83?SVM?.The results showed that hsa_circ₀005045 and hsa_circ₀037516 may had an potential interaction binding site with PRDX2.In brief,we found that hsa_circ₀005045 was significantly up-regulated in plasma of COPD patients exposed to PM_2.5,which was verified at the cellular and animal levels.hsa_circ₀005045 may bind to exosomal protein PRDX2 and ELANE,therefore participate in the process of PM_2.5 induced COPD.Our study suggested that hsa_circ₀005045 can be used as a new molecular marker for the development of COPD related to PM_2.5 exposure,providing a new perspective for the intervention target of respiratory injury caused by PM_2.5.